Design and synthesis of triazolopyrimidine acylsulfonamides as novel anti-mycobacterial leads acting through inhibition of acetohydroxyacid synthase

Article abstract of DOI:10.1016/j.bmcl.2014.02.054

Novel triazolopyrimidine acylsulfonamides class of antimycobacterial agents, which are mycobacterial acetohydroxyacid synthase (AHAS) inhibitors were designed by hybridization of known AHAS inhibitors such as sulfonyl urea and triazolopyrimidine sulfonamides. This Letter describes the synthesis and SAR studies of this class of molecules by variation of two parts of the molecule, the phenyl and triazolopyrimidine rings. SAR study describes optimisation of enzyme potency, whole cell potency and evidence of mechanism of action.

Full text of DOI:10.1016/j.bmcl.2014.02.054

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of triazolopyrimidine acylsulfonamides
as novel anti-mycobacterial leads acting through inhibition
of acetohydroxyacid synthase
Vikas Patil a, Manoj Kale a, Anandkumar Raichurkar a, Brahatheeswaran Bhaskar c, Dwarakanath Prahlad a,
Meenakshi Balganesh a, Santosh Nandan b, P. Shahul Hameed a,
⇑
a AstraZeneca India Pvt. Ltd, Avishkar, Kirloskar Business Park, Bellary Road, Hebbal, Bangalore 560 024, Karnataka, India
b Ambernath Organics, 307/314, Creative Industries Premises, Road No. 2, Sunder Nagar, Kalina, Mumbai 400 098, India
c EDC Creative Technology Solutions Private Limited, EDC Conclave, Jeevith Gardens [Off. ITPL Road], Bangalore 560 037, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel triazolopyrimidine acylsulfonamides class of antimycobacterial agents, which are mycobacterial
acetohydroxyacid synthase (AHAS) inhibitors were designed by hybridization of known AHAS inhibitors
such as sulfonyl urea and triazolopyrimidine sulfonamides. This Letter describes the synthesis and SAR
studies of this class of molecules by variation of two parts of the molecule, the phenyl and triazolopyr-
imidine rings. SAR study describes optimisation of enzyme potency, whole cell potency and evidence
of mechanism of action.
Received 15 November 2013
Revised 28 January 2014
Accepted 18 February 2014
Available online xxxx
Keywords:
Ó 2014 Elsevier Ltd. All rights reserved.
Triazolopyrimidine acylsulfonamides
Acetohydroxyacid synthase
Antimycobacterial
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb),
is responsible for over 1.4 million deaths annually and 9 million
new cases of infection each year. The re-emergence of TB due to
HIV co-infection, multi-drug resistant (MDR-TB) and extremely
drug resistant (XDR-TB) strains of Mtb has created a global epi-
demic with serious consequences if left unaddressed.1 The resolu-
tion of the current TB epidemic requires not only prevention of
new infections but also new medicines that are safe and effective
against drug sensitive and drug resistant Mtb strains.2
It is well known in the literature that drugs that act via novel
mechanisms of action would be effective against both drug sensi-
tive as well as drug resistant TB and could also potentially help
to replace one or more of the current drugs that exhibit severe side
effects.
2-acetolactate, or with a molecule of 2-ketobutyrate to yield
2-aceto-2-hydroxybutyrate, common precursors for the synthesis
of all three branched chain amino acids. Since mycobacteria synthe-
size all the amino acids required for their protein synthesis, the
amino-acid biosynthesis pathways are essential for their survival.
Such BCAA pathway are absent in humans.7 As AHAS is also an
essential enzyme in plants it has been successfully targeted for
developing novel herbicides. Commercially marketed herbicides
such as sulfonylurea sulfometuron methyl (SMM)8 and triazolo-
pyrimidine sulfonamides9a are known to be potent inhibitors of
plant AHAS (Fig. 1).
Interestingly, SMM has been shown to inhibit the growth of Mtb
and shown to be efficacious in a murine mouse model, albeit at
In the recent literature, acetohydroxyacid synthase (AHAS) has
been recognized as an attractive enzyme target for discovering novel
anti-TB compounds.3–6 AHAS is the first enzyme in the pathway for
the de novo biosynthesis of branched chain amino acids [BCAA]
isoleucine, leucine and valine (ilv). AHAS catalyses the irreversible
decarboxylation of pyruvate and the condensation of the
acetaldehyde moiety with a second molecule of pyruvate to give
CH3
O
CH3
N
N
N
N
H3C
O
NH
S
O
O
O
H3C
N
O
R1
S
H3C
N
N
H
N
H
O
R2
Sulfometuron Methyl (SMM)
Triazolopyrimidine Sulfonamides
Flumetasulam, wherein R1= 1-F and R2 = F
⇑
Corresponding author. Tel.: +91 8042188361.
E-mail addresses: shahul.mehar@astrazeneca.com, shahulmehar@yahoo.com
Figure 1. Plant AHAS inhibitors as herbicides.
(P. Shahul Hameed).
http://dx.doi.org/10.1016/j.bmcl.2014.02.054
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Patil, V.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.02.054

2
V. Patil et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
higher dose (500 mg/kg, ip), suggesting that AHAS is an essential
target for the survival of Mtb.3
However, recent studies have shown that inactivation of ilvB1
(coding for AHAS large subunit) gene in Mtb leads to BCAA auxot-
rophy and attenuation of virulence in mice. This may be due to
either uptake of BCAA from in vivo environment or ilvB1 mutant
having ability to synthesize some BCAA using an alternative mech-
anism, or combination of both.10
Based on the above studies, one would assume that inhibiting
AHAS in Mtb with a small molecule inhibitor would result in bac-
teriostatic effect in vivo and not bactericidal, such compounds
when given in combination with other anti-tuberculosis drugs
may lead to better efficacy. In fact, we have recently demonstrated
that rifampicin, a potent RNA polymerase inhibitor and one of the
front-line TB drugs, potentiates the killing effects of several of the
AHAS inhibitors in vitro.11 Therefore, the focus of the current study
was to identify potent Mtb specific AHAS inhibitors with improved
whole cell potency against Mtb.
Figure 3. Overlay of Flumetasulam (magenta) and compound 3d (yellow) onto
crystal structure (1YI0) bound conformation of sulfometuron (green).
Our approach towards designing AHAS inhibitors with im-
proved whole cell potency was to utilize the structures 1 and 2
as starting points. We hypothesized that hybridization of structural
features of sulfonylureas and triazolopyrimidine sulfonamides
would result in compounds such as triazolopyrimidines acylsulf-
onamide (3) that may maintain overall structural features required
to bind to the bacterial enzyme through the critical residues
(Fig. 2). The triazolopyrimidine acylsulfonamides 3 are also already
known in literature as herbicides or plant growth regulants.12,13
It is interesting to note here that the genesis of triazolopyrimi-
dine sulfonamides itself has its origin in the conformational
restriction of sulfonylureas (Fig. 3).9a Overlay of SMM (crystal
bound conformation as in 1YI0),9b triazolopyrimidine sulfonamide
(Flumetasulam) and triazolopyrimidine acylsulfonamide 3d is de-
picted in Fig.4.
As evident from the overlay, the common feature present in
both sulfonylureas and triazolopyrimidine sulfonamides is the
presence of SO2NH group. The carbonyl group of 3d occupies the
same region as that of carbonyl group present in SMM and allows
the pyrimidine part of triazolopyrimidine ring (3d) to come closer
to same position as that of pyrimidine ring of SMM.
To validate our hypothesis, we evaluated the binding mode of
SMM and compound 3d through docking using Mtb AHAS homol-
ogy model built over plant AHAS (1YI0) as a template. Docking
studies (Fig. 4) suggest that the binding mode and the observed
interactions of SMM and 3d were quite similar to sulfonylureas
class of compounds, reported in the crystal structure of plant and
yeast AHAS.9b,c The carbonyl of 3d and one of the nitrogen atom
of triazolopyrimidine ring forms crucial hydrogen bond (HB) inter-
actions with terminal nitrogen’s of conserved Arg377 mimicking
carbonyl and pyrimidine ring nitrogen of SMM. The sulfonyl
oxygen of 3d forms HB interaction with Lys197 whereas the
Figure 4. Comparison of Hypothetical binding modes of SMM (stick model, green)
and compound 3d (stick mode, yellow) in Mtb AHAS homology model (grey,
important residues are shown in stick model). Dash line indicates hydrogen bond
interactions.
triazolopyrimidine ring of 3d forms a very strong
p–p stacking
interaction with indolyl group of Trp516. Thus, the newly designed
triazolopyrimidine acylsulfonamide class of compounds may act as
novel Mtb AHAS inhibitors.
Coincidentally acylsulfonamides, such as 4 and 5, have been re-
ported to be inhibitors of plant AHAS14 thus giving credence to our
thinking that bioisosteric replacement of sulfonamide with acyl-
sulfonamides may be an valid strategy to identify new inhibitors
of Mtb AHAS (Fig. 5).
We have designed and synthesized differently substituted triaz-
olopyrimidine acylsulfonamide class of compounds with variations
in the phenyl sulfonamide part and tested. Here, we present AHAS
enzyme inhibition and MIC data for triazolopyrimidine acylsulf-
onamides in Table 1.
The title series of triazolopyrimidine acylsulfonamides were
synthesized in a convergent fashion by coupling sulfonamides 11
and triazolopyrimidine carboxylic acid 9 using EDCI as coupling re-
agent (Scheme1). 9 was obtained by condensing ethyl 5-amino-
4H-1,2,4-triazole-3-carboxylate (6) with acetylacetone (7) as per
the literature procedure.12 The sulfonamides used were either
commercially available or were conveniently synthesized by
R1
CH3
N
O
O
S
H3C
N
N
H
N
R2
O
H
CH3
N
O
N
N
R1
Sulf onyl Urea (1)
O
S
CH3
HN
H3C
N
O
N
N
R2
(3)
NH
O
O
R2
H
N
O
N
Hybrid Molecule
Triazolopyrimidine acylsulfonamides
O
S
H3C
N
H
N
N
O
R1
S
N
H
S
Ar
O
Ar
O
O
O
Triazolopyrimidine sulfonamides (2)
Figure 2. Hybridization approach to get triazolopyrimidines acylsulfonamide.
4
5
Figure 5. Acylsulfonamides as AHAS inhibitors.
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V. Patil et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
Table 1
Mtb AHAS pIC50, Mtb MIC and Mtb MIC in presence of ilv
a
b
Compound
R1
R2
Mtb AHAS pIC50
Mtb MICc
(
lg/ml)
Mtb MIC+200 (l
g/ml) ilvd
SMM
4
3a
3b
3c
3d
3e
—
—
o-CF3
o-Cl
o-Br
—
—
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
6.12
<4.52
6.98
6.82
7.04
7.15
7.60
5.24
6.29
5.81
>7.2
6.59
5.31
5.36
5.60
<4.52
<4.52
6.70
7.75
6.54
16
>32
NDe
>32
>32
>32
>32
>32
>32
>32
>32
>32
ND
>32
60.13
2
0.25
0.25
0.03
>32
1
0.5
<0.25
32
32
16
o-COOMe
o-OCF3
o-F
o-CH3
o-OCH3
o-OCHF2
o-CH2CN
m-Br
m-CF3
m-OCF3
p-OCF3
p-CF3
o-Cl
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3ab
3ae
3ag
ND
ND
ND
ND
16
>32
>32
0.25
60.03
0.5
ND
Me
Me
Me
>32
>32
>32
o-OCF3
o-CH3
a
b
c
Mtb AHAS: Mycobacterium tuberculosis Acetohydroxyacid synthase.
pIC50: –log IC50 (Mtb AHAS inhibition).
MIC: Minimum Inhibitory Concentration (unit as given).
ilv : Isoleucine, Leucine and Valine.
d
e
ND: Not determined.
may lead to twisting of the phenyl ring with respect to the rest
of the molecule. Based on this data, we synthesized a few 2,6 di-
substituted sulfonamides (3ab, 3ae, 3ag) with methyl group at R2
position. This result suggested that twisting of the aryl ring may
not be an important determinant for activity of this class of mole-
cules and in-fact it had potentiated the AHAS inhibition for 2,6 di-
substituted compounds.
The data in Table 1 shows that compounds 3a–3ag with varied
enzyme inhibition potencies showed moderate to potent Mtb MIC.
In general, the cellular potency (MIC) fairly correlates with enzyme
potency (pIC50) for this series. A closer look at the data suggested
CH3
N
N
CH3
O
O
i (a,b)
O
OH
O
N
N
+
N
H2N
N
H
H3C
CH3
O
H3C
N
6
7
9
R1
R1
O
iii
Cl
NH2
O
ii
S
S
O
R2
R2
O
CH3
N
10 (a-ag)
11 (a-ag)
O
R2
N
O
that the MICs did not correlate well with enzyme inhibition (pIC50
)
N
HN
S
O
H3C
N
for few compounds (e.g. 3b and 3h). This could be due to differential
bacterial permeability or efflux property of the compounds resulting
in moderate correlation between pIC50 and MIC for these com-
pounds. It is interesting to note that additional methyl substitution
at R2 position not only potentiated the enzyme potency but also im-
proved MIC as compared to its mono substitutedcounterpart (e.g. 3b
vs 3ab). This pattern is structurally consistent with all 2,6 di-substi-
tuted compounds (3ab, 3ab, and 3ag). Furthermore, when
compounds were tested in the presence of ilv, the MICs were ele-
vated suggesting that growth inhibition is due to the amino-acids
starvation and not by any other mechanism and that the target is
auxotrophic in nature.
In conclusion, we have demonstrated triazolopyrimidine acyl-
sulfonamides as new leads with potent anti-TB activity acting
through the inhibition of Mtb AHAS enzyme. The molecules exhib-
ited potent Mtb AHAS inhibition as well as Mtb MIC via the ex-
pected mechanism of action and were more potent than
sulfonylureas and triazolopyrimidine sulfonamides reported
earlier.
R1
3 (a-ag)
Scheme 1. Reagents and conditions: (i) (a) Piperidine/EtOH, 100 °C, reflux, over-
night (b) 2.5 N NaOH/EtOH (ii) CH3CN/EDCI, DMAP, 50 °C 3 h (iii) aq NH3/CH3CN,
0 °C to rt, 2 h.
converting respective sulfonyl chlorides (10) to sulfonamides using
aqueous ammonia.
In addition to these molecules, SMM was also tested to establish
the baseline. The acylsulfonamide 4 was found to be inactive
against Mtb AHAS but the newly designed acylsulfonamides
(3a–3ai) showed potent activity in the primary enzyme assay as
shown in Table 1.
As shown in Table 1, the ortho substituted analogs (3a–3j)
showed potent AHAS inhibition (pIC50 from 5.24 to >7.2), whereas,
the meta-substituted analogs (3k–3m) were moderately active
with pIC50 in the range of (5.3–5.6) and the para substituted ana-
logs (3n and 3o) were inactive. Although electron withdrawing
groups in the ortho position of phenyl ring seems to be favoured,
this is not always the case as seen with compounds 3g and 3h,
which have electron donating groups. Analysis of SAR reveals that
the inhibition could be ascribed to electron withdrawing nature as
well as size of ortho substitution of phenyl ring. The moderate
activity of the methoxy and methyl groups (3g, 3h) reinforces
the fact that both electronics as well as steric factor plays an
important role in bringing AHAS inhibition.
These molecules are uniquely placed to serve as leads, as there
are sufficient structural handles that can be explored within the
triazolopyrimidine ring for further optimization to identify a candi-
date drug with the potential to synergize with a RNA polymerase
inhibitor such as Rifampicin.
Acknowledgments
We deeply acknowledge the analytical support provided
by Suresh Rudrapatna and Lavakumar Naviri. We also thank
The presence of another ortho substituent in the phenyl portion
of triazolopyrimidine acylsulfonamide, such as a methyl group
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4
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